EP2893041A2 - Anticorps chimère anti-adndb/anti-chromatine - Google Patents

Anticorps chimère anti-adndb/anti-chromatine

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Publication number
EP2893041A2
EP2893041A2 EP13834463.5A EP13834463A EP2893041A2 EP 2893041 A2 EP2893041 A2 EP 2893041A2 EP 13834463 A EP13834463 A EP 13834463A EP 2893041 A2 EP2893041 A2 EP 2893041A2
Authority
EP
European Patent Office
Prior art keywords
antibody
chimeric antibody
chromatin
dsdna
chimeric
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP13834463.5A
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German (de)
English (en)
Other versions
EP2893041A4 (fr
Inventor
Roger WALKTER
John Wesley BRENEMAN, III
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bio Rad Laboratories Inc
Original Assignee
Bio Rad Laboratories Inc
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Filing date
Publication date
Application filed by Bio Rad Laboratories Inc filed Critical Bio Rad Laboratories Inc
Publication of EP2893041A2 publication Critical patent/EP2893041A2/fr
Publication of EP2893041A4 publication Critical patent/EP2893041A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • Systemic autoimmune disorders are characterized by circulating autoantibodies to certain antigens, such as those present in the cell nucleus.
  • Antinuclear antibodies can also be indicative of viral and bacterial infections, hypertension, cancers, and psoriasis.
  • the calibration antibody is similar to the native autoantibody to be detected so that assay conditions are kept as constant as possible, and the concentration determination is accurate. For this reason, present methods often utilize autoantibodies from native sources. Such autoantibodies, however, do not have predictable properties and are rare, so that obtaining a reliable source of calibration antibody is difficult and costly.
  • antibodies e.g., chimeric antibodies or single chain antibodies
  • the presently disclosed antibodies have similar properties as the native autoantibodies to be detected, and are easily and predictably produced.
  • the constant region is derived from a human antibody.
  • the constant region and framework regions are derived from a human antibody.
  • the antibody isotype can be IgG (IgGl , IgG2, IgG3, IgG4), IgM, IgA, IgE, or IgD.
  • the chimeric antibody comprises complementarity determining regions (CDRs) derived from a non-human animal.
  • CDRs complementarity determining regions
  • chimeric antibody comprises a variable region derived from a non-human animal.
  • the non-human animal is selected from a rodent (mouse, rat, hamster), rabbit, horse, goat, pig, sheep, chicken, and bovine.
  • the anti-dsDNA/chromatin antibody is stable at 5°C for at feast 5 months, e.g., at least any one of 6, 9, 12, 15, 18, 21 , or 24 months. In some embodiments, the anti-dsDNA/chromatin antibody is stable at for about the same duration (e.g. , ⁇ about 2, 5, or 10%) as a native human antibody that specifically binds dsDNA or chromatin in given conditions (e.g., temperature, buffer).
  • the anti-dsDNA'chromatin antibody is labeled, either directly or indirectly (e.g., with a secondary antibody or other indirect method such as Protein A, G, A/G, or strep-bio).
  • the anti-dsDNA/chromatin antibody is recognized by (specifically bound by) a labeled secondary antibody.
  • the secondary antibody is specific for human antibodies (anti-human).
  • both the anti-dsDNA/chromatin antibody and secondary antibody are labeled, e.g., with different labels.
  • the label is fluorescent.
  • the anti-dsDNA/chromatin antibody specifically binds human dsDNA and human chromatin.
  • the chimeric anti- dsDNA/chromatin antibody specifically binds dsDNA in a non-sequence specific manner.
  • the anti-dsDNA/chromatin antibody has a linear dilution profile within a range of 10-9000 relative fluorescence intensity (RFI), 100-9000 RFI, 1000- 9000 RFI, 10-5000 RFI, 100-2500 RFI, or 50-5000 RFI for dsDNA.
  • the anti-dsDNA/chromatin antibody has a linear dilution profile within a range of 10-1500 RFI, 10-1000 RFI, 50-1000 RFI, 100-1500 RFI, 10-500 RFI, or 50-500 RFI for chromatin.
  • the anti-dsDNA/chromatin antibody is in a solution with at least one additional antibody specific for a different target, e.g., a different nuclear antigen target.
  • a different target e.g., a different nuclear antigen target.
  • ihe solution comprises an anti-dsDNA/'chromatin antibody as described herein and at least one additional antibody that specifically binds a target (antigen) from the cell nucleus or nucleolus, e.g., an antigen selected from the group consisting of: ribosomal protein, SS-A52, SS-A60, SS-B, Sm, Sm/ribonuclear protein (RNP), RNP-A, RNP-68, Scl-70, Jo- 1, and ceniromere B.
  • ribosomal protein SS-A52, SS-A60, SS-B, Sm, Sm/ribonuclear protein (RNP), RNP-A, RNP-68, Scl-70, Jo-
  • kits for determining the amount of a sample (test) antibody that specifically binds dsDNA and/or chromatin comprising at least one container with a defined (known) amount of anti-dsDMA/chromatin antibody.
  • the sample antibody is in or is obtained from a biological sample from a human.
  • the kit includes a container for the sample antibody and optionally a device for obtaining the biological sample.
  • the kit includes a labeled secondary- antibody, e.g., an anti-human secondary antibody.
  • the anti- dsDNA/chromatin antibody is labeled, e.g., with a different label than the secondary antibody label.
  • the kit comprises two or more containers comprising the anti- dsDN 'chromatin antibody, wherein each of the two or more containers holds a different defined (known) amount of the anti-dsD A/chromatin antibody.
  • the kit further comprises at least one container of dsDNA and/or chromatin, e.g., in a known amount.
  • the method comprises contacting an anti-dsDNA/chromatin antibody as described herein at a first known amount with dsDNA or chromatin in a first solution, detecting binding of the anti- dsDNA/chromatin antibody to the dsDNA or chromatin, and assigning a detection value to the first known amount; contacting the anti-dsDNA/chromatin antibody at a second known amount with dsDNA or chromatin in a second solution, wherein the dsDNA or chromatin is present at the same amount in the first and second solutions, detecting the binding of the anti- dsDIsl A'chromatin antibody to the dsDNA or chromatin, and assigning a second detection value to the second known amount of anti-dsDNA/chromatin antibody, thereby generating a calibration curve of the anti-dsDNA/chromatin antibody.
  • the method comprises contacting the anti-dsDNA/chromatin antibody with dsDNA, and the known amounts of anti-dsDNA/chromatin antibody are in the range of 0.001 to 100 ug/mL, e.g., 0.01 to 20 ug/mL, 0.01 to 5 ug/mL or about 0.05 to 0.5 ug mL.
  • the method comprises contacting the anti-dsDNA/chromatm antibody with chromatin, and the known amounts of anti-dsDNA/chromatin antibody are in the range of 0,01 to 0.5 ug/mL.
  • the method further comprises repeating the steps of contacting, detecting, and assigning additional detection values for additional known, amounts of anti-dsDNA/chromatin antibody, and comparing the detection value of the sample antibody to the additional detection values, wherem the dsDNA or chromatin is present at the same amount in each of the solutions.
  • the steps of contacting, detecting, and assigning detection values are repeated for 3, 4, 5, 6, 7, 8, 9, or 10 known amounts of the anti-dsDNA/chromatin antibody.
  • the dsDNA or chromatin is attached to a substrate (e.g.
  • the detecting comprises contacting the chimeric anti- dsDNA/chromatin antibody and/or sample antibody with a labeled secondary antibody.
  • the same secondary antibody is used to detect both the chimeric anti- dsD A/chromatin antibody and the sample antibody.
  • the method comprises contacting the anti-dsDNA/chromatin antibody and sample antibody with dsDNA, and the known amounts of anti- dsDNA/chromatin antibody are in the range of 0.01 to 10 ug/mL. In some embodiments, the method comprises contacting the anti-dsDNA/chromatin antibody and sample antibody with chromatin, and the known amounts of anti-dsDNA/chromatin antibody are in the range of 0.01 to 0.5 ug/mL.
  • the sample antibody is obtained from or is in a biological sample from a human.
  • the method further comprises determining whether the human has an autoimmune disorder based on the amount or presence of the sample antibody.
  • the method can comprise diagnosing an autoimmune disorder in the human w r here the sample antibody is detected.
  • the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, mixed connective tissue disease, Sjogren's syndrome, scleroderma, dermatomyositis, polymyositis, and CREST syndrome, rheumatoid arthritis, juvenile arthritis, and Felly's syndrome.
  • Figure I is a standard depiction of a tetrameric antibody structure with two light chains and two heavy chains.
  • the variable region is shown as the top portion of each chain.
  • the antibody on the right is chimeric, with a variable region derived from a mouse antibody and a constant region derived from a human antibody
  • Figure 2 shows stability of antibody binding to dsDNA at 25°C over 14 days (equivalent to 5.6 months at 5°C).
  • A compares the signal retained versus day 0 signal for each antibody
  • B compares signal retained versus day -matched 5°C signal for each antibody.
  • Positive control antibodies derived from native antibodies specific for dsRIS!A and chromatin, are designated Calib L04, LOS, and L06.
  • GG 1 :50 refers to Glycine-HCL Glycerol eluted Chimeric antibody clone 20, at a 1 :50 dilution
  • GG 1 : 100 refers to Glycine- HCL Glycerol eluted Chimeric antibody clone 20, at a 1 : 100 dilution
  • GT 1 :25 refers to Glycyltyrosine eluted Chimeric antibody clone 20, at a 1 :25 dilution.
  • Figure 3 shows stability of antibody binding to dsDNA at 37°C over 14 days (equivalent to 20.5 months at 5°C).
  • A compares the signal retained versus day 0 signal for each antibody
  • B compares signal retained versus day -matched 5°C signal for each antibody.
  • the antibodies are designated as in Figure 2.
  • Figure 4 shows stability of antibody binding to chromatin at 25°C over 14 days (equivalent to 5.6 months at 5°C).
  • A compares the signal retained versus day 0 signal for each antibody
  • B compares signal retained versus day -matched 5°C signal for each antibody.
  • the antibodies are designated as in Figure 2,
  • Figure 5 shows stability of antibody binding to chromatin at 37°C over 14 lays (equivalent to 20.5 months at 5°C).
  • A compares the signal retained versus day 0 signal for each antibody.
  • B compares signal retained versus day-matched 5°C signal for each antibody.
  • the antibodies are designated as in Figure 2.
  • Figure 6 shows linear signal to concentration relationship for chimeric antibody clones 20, 22, and 28. Plateau for clone 20 is due to detector limit. RFI: relative fluorescence intensity.
  • Figure 7 shows linear signal (dsDNA) to concentration relationship for chimeric antibody clones 20, 22, and 28.
  • RFI relative fluorescence intensity.
  • Figure 8 shows linear signal (chomatin) to concentration relationship for chimeric antibody clones 20, 22, and 28.
  • RFI relative fluorescence intensity
  • Figure 9 shows linear signal from both dsDNA and chromatin with chimeric antibody clone 20. Note that the typical assay range for dsDNA is 10-9000 RFI (0-300 lU/mL), while it is 10- 1500 RFI (0-8 AI (antibody index)) for chromatin.
  • chimeric monoclonal antibodies that specifically bind to dsDNA and chromatin.
  • Naturally-occurring antibodies specific for dsDNA and/ or chromatin are typically not found in high concentrations, and have variable binding characteristics (e.g., affinity, avidity, epitope, etc.).
  • the presently described antibodies can be clonally or recombinantly expressed, thus providing a reliable source of antibodies with known binding characteristics.
  • the presently described antibodies are stable in storage and assay conditions, and can detect dsDNA and chromatin in the same linear concentration range.
  • autoantibody refers to an antibody produced by an individual that specifically binds an epitope in the same individual. Autoantibodies can be described as directed against "self” antigens, and can be indicative of an autoimmune disease. For example, individuals with multiple sclerosis produce autoantibodies that specifically bind a component of the myelin sheath that normally protects nerve cells. Autoantibody binding in MS patients results in recruitment of immune cells that damage and degrade the myelin, and subsequent damage to the underlying nerve cells.
  • ANA Anti-Nuclear Antibody
  • ANA refers to an antibody that specifically binds a substance normally found in a cell nucleus, e.g., dsDNA, chromatin, ribosomal proteins, centromeric proteins (e.g.. Centromere B), SS-A, SS-B, Sm, Sm/RNP, RNP, Scl-70, Jo- 1 , etc.
  • the presence of ANAs that are also autoantibodies in an individual can be indicative of particular autoimmune conditions, e.g.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof.
  • polynucleotide refers to a linear sequence of nucleotides.
  • nucleotide typically refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA (including siRNA), and hybrid molecules having mixtures of single and double stranded DN A and RNA..
  • double stranded DNA or "dsDNA” is intended to refer to a
  • DN A strand deoxyribonucleotide polymer hybridized to its complement through Watson- Crick bonding.
  • the dsDNA can be of any length and can be associated with additional components (e.g., histone proteins or proteins involved in replication or transcription).
  • additional components e.g., histone proteins or proteins involved in replication or transcription.
  • the two strands of DNA may not be 100% complementary, so long as the percentage is high enough in the given conditions for the two strands to remain associated.
  • sequence A-G-T is complementary to the sequence T-C-A.
  • Complementarity may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where ail the nucleic acids match according to base pairing.
  • eiecrrophoretic separation e.g., Southern blot for detecting DNA, and Northern blot for detecting RNA
  • measurement of DNA and RNA can also be carried out in the absence of eiecrrophoretic separation (e.g., quantitative PGR, dot blot, or array).
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y- carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical stmcture as a naturally occurring amino acid, e.g., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated, e.g., naturally contiguous, sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations.
  • Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan.
  • TGG which is ordinarily the only codon for tryptophan.
  • the following amino acids are typical ly conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamme (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T j; and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins ( ⁇ 9M)).
  • nucleic acids in the context of two or more nucleic acids, or two or more polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides, or amino acids, that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters, or by manual alignment and visual inspection.
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the celi is derived from a celi so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the ceil or express native genes that are otherwise abnormally expressed, under expressed or not expressed at ail
  • the term “native” or “naturally occurring” refers to a substance (e.g., protein, antibody, nucleic acid) that is not modified from its natural form. A native or naturally occurring substance can, however, be isolated from its natural environment.
  • the term “primary antibody” will be understood by one of skill to refer to an antibody or fragment thereof that specifically binds to an analyte (e.g., substance, antigen, component) of interest.
  • the primary antibody can further comprise a tag, e.g., for recognition by a secondary antibody or associated binding protein (e.g., GFP, biotin, or strepavidin), or to facilitate separation (e.g., a poly-His tag), 10051]
  • a secondary antibody or associated binding protein e.g., GFP, biotin, or strepavidin
  • the term "secondary antibody” refers to an antibody that specifically binds to a primary antibody.
  • a secondary antibody can be specific for the primary antibody (e.g., specific for primary antibodies derived from a particular species) or a tag on the primary antibody (e.g., GFP, biotin, or strepavidin).
  • Secondary antibodies are usually attached to a detectable moiety or a matrix for separation (e.g., a bead, chromatography agent, array, or ELISA plate).
  • the term includes but is not limited to polyclonal or monoclona l antibodies of the isoiype classes IgA, IgD, IgE, IgG, and TgM, derived from human or other mammalian cells, including natural or genetically modified forms such as humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies.
  • a exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-ierminus of each chain defines a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (V3 ⁇ 4) refer to these light and heavy chains respectively.
  • the variable region contains the antigen- binding region of the antibody (or its functional equivalent) and is most critical in specificity and affinity of binding. See Paul, Fundamental Immunology (2003).
  • Antibodies can exist as intact immunoglobulins or as any of a number of well- characterized fragments that include specific antigen-binding activity. Such fragments can be produced by digestion with various peptidases. Pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The Fi ab f > may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)' 2 dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region.
  • antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology.
  • antibody also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies or those identified using phage display libraries (see, e.g., McCafferry et at, Nature 348:552-554 (1990)).
  • variable region fragment refers to a monovalent or bi-valent variable region fragment, and can encompass only the variable regions (e.g., VL and/or ' ⁇ ), as well as longer fragments, e.g., an Fab, Fab' or F(ab')2, which also includes C-L and/or CRI - Unless otherwise specified, the term “Fc” refers to a heavy chain monomer or dimer comprising (3 ⁇ 41 and CH2 regions.
  • a single chain Fv refers to a polypeptide comprising a VL and V H joined by a linker, e.g., a peptide linker.
  • ScFvs can also be used to form tandem (or di-valent) scFvs or diabodies. Production and properties of tandem scFvs and diabodies are described, e.g., in Asano et al. (201 X) J Biol. Chem. 286: 1812; Kenanova et al. (2010) Prot Eng Design Sel 23:789; Asano et al. (2008) Prot Eng Design Sel 21 :597.
  • A. "monoclonal antibody” refers to a clonal preparation of antibodies with a single binding specificity and affinity for a given epitope on an antigen
  • a “polyclonal antibody” refers to a preparation of antibodies that are raised against a single antigen, but that includes antibodies with different binding specificities and affinities for epitopes on the single antigen.
  • CDR complementarity-determining region
  • V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found
  • VL CDRl is the CDR from the variable domain of the light chain of the antibody in which it is found.
  • the sequences of the framework regions of different fight or heavy chains are relatively conserved within a species.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDR s in three dimensional space.
  • amino acid sequences of the CDRs and framework regions can be determined using various well known definitions in the art, e.g., Kabat, Chothia, international
  • ImMunoGeneTics database IMGT
  • AbM ImMunoGeneTics database
  • IMGT ImMunoGeneTics database
  • AbM AbM
  • a helpful guide for locating CDRs using the Kabat system can be found at the website available at bioinf.org.uk/abs. Definitions of antigen combining sites are also described in the following: Ruiz et al.
  • chimeric antibody refers to an antibody in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region, CDR, or portion thereof) is linked to a constant region of a different or altered class, effector function and/or species; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity (e.g. , CDR and framework regions from different species).
  • Chimeric antibodies can include variable region fragments, e.g., a recombinant antibody comprising two Fab or Fv regions or an scFv.
  • a chimeric antibody can also, as indicated above, include an Fc region from a different source than the attached Fv regions. In some cases, the chimeric antibody includes chimerism within the Fv region.
  • An example of such a chimeric antibody would be a humanized antibody where the FRs and CDRs are from different sources.
  • antigen a molecule, compound, or complex that is recognized by an antibody, i.e., can be specifically bound by the antibody.
  • the term can refer to any molecule that can be specifically recognized by an antibody, e.g., a polynucleotide, polypeptide, carbohydrate, lipid, chemical moiety, or combinations thereof (e.g., phosphorylated or glycosylated polypeptides, etc.).
  • a polynucleotide polypeptide
  • carbohydrate e.g., lipid, chemical moiety, or combinations thereof (e.g., phosphorylated or glycosylated polypeptides, etc.).
  • Antibodies bind to an "epitope" on an antigen.
  • the epitope is the localized site on the antigen that is recognized and bound by the antibody.
  • Protein epitopes can include a few amino acids or portions of a few amino acids, e.g., 5 or 6, or more, e.g., 20 or more amino acids, or portions of those amino acids.
  • Epitopes can also include non-protein components, e.g. , nucleic acid (e.g., RNA or DNA), carbohydrate, or lipid. Epitopes can also include combinations of these components. In some cases, the epitope is a three-dimensional moiety.
  • the term "binds" with respect to an antibody target typically indicates ihai an antibody binds a majority of the antibody targets in a pure population, assuming an appropriate molar ratio of antibody to target.
  • an antibody that binds a given antibody target typically binds to at least 2/3 of the antibody targets in a solution (e.g., 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%).
  • a solution e.g., 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.
  • a first antibody, or an antigen-binding portion thereof "competes" for binding to a target with a second antibody, or an antigen-binding portion thereof, when binding of the second antibody with the target is detectably decreased in the presence of the first antibody compared to the binding of the second antibody in the a bsence of the first antibody.
  • the reverse, where the binding of the first antibody to the target is also detectably decreased in the presence of the second antibody can exist, but need not be the case. That is, a second antibody can inhibit the binding of a first antibody to the target without that first antibody inhibiting the binding of the second antibody to the target.
  • competitive antibody can be applied to the first or second antibody as can be determined by one of skill in the art, in some cases, the presence of the competitor antibody (e.g., the first antibody) reduces binding of the second antibody to the target by at least 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more, e.g., so that binding of the second antibody to target is undetectable in the presence of the first (competitor) antibody.
  • label refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • useful labels include fluorescent dyes, luminescent agents, radioisotopes (e.g., ,2 P, ' ⁇ ), electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radioiabel into a peptide or antibody specifically reactive with a target analyte. Any method known in the art for conjugating an antibody to the label may be employed, e.g., using methods described in Hermanson,
  • tag can be used synonymously with the term “label,” but generally refers to an affinity- based moiety, e.g. , a "His tag” for purification, or a “strepavidin tag” that interacts with biotin.
  • a "labeled" molecule e.g., nucleic acid, protein, or antibody
  • a "labeled” molecule is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der W aals, electrostatic, or hydrogen bonds to a label such that the presence of the molecule may be detected by detecting the presence of the label bound to the molecule.
  • a control can be devised to compare signal strength in given conditions, e.g., in the presence of a test antibody, in the absence of the test antibody (negative control), or in the presence of a known antibody with a known affinity (positive control).
  • a control can be devised to compare signal strength in given conditions, e.g., in the presence of a test antibody, in the absence of the test antibody (negative control), or in the presence of a known antibody with a known affinity (positive control).
  • controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.
  • stable indicates that the antibody retains a certain level of activity at given conditions (e.g., temperature, duration, pFi, etc.). Activity can be expressed in terms of target binding (e.g., in terms of amount of target bound). Thus, an antibody can be considered stable if it retains at least any of 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or higher target binding compared to a control.
  • target binding e.g., in terms of amount of target bound
  • an antibody activity, and stability can be expressed using other criteria, e.g. , structural criteria, target binding affinity, etc. The stability of the antibody can be considered with relation to time, so that antibody activity at a starting time is compared to activity at later times.
  • the stability can also be considered in different buffer conditions, different states (e.g. , pre- and post-lyophilization, pre- and post- freezing) or at different temperatures (e.g. , activity at a control temperature compared to higher or lower temperatures).
  • the "amount" of a substance e.g., antibody, target molecule, protein, nucleic acid, etc.
  • the amount of chromatin can be expressed according to Antibody Index (AI), an arbitrary comparative measure.
  • the amount of dsDNA can be expressed using International Units (III). Amounts can be expressed in terms of relative fluorescent intensity (RFI), in terms of concentration (e.g., mg/ml or molarity), according to mass or binding units, etc.
  • a calibration curve is a tool for determining the amount or concentration of a substance in a sample by comparing the uniaiown amount as detecied to a set of standards of known amounts.
  • Calibration curves reveal the limit of detection (LOD) and limit of linearity (LOL) for a given assay, in the context of the present disclosure, the substance of unknown amount can be an autoantibody from a patient sample, and the calibration standards are known amounts of an antibody specific for the same antigen.
  • LOD limit of detection
  • LEL limit of linearity
  • a “linear dilution profile,” as used herein, indicates that antibody activity (e.g., target binding) correlates with its concentration in a linear manner.
  • the calibration "curve" can be linear.
  • the chimeric anti-dsD A/chromatin antibodies described herein can be used with any antibody-based assay or separation procedure, and are conveniently used as a standard for determining the amount of or binding ability of a test antibody.
  • the known target and binding ability of the presently described antibodies can be used as a baseline comparison.
  • Antibody binding to a target can be detected using immunoassays, for example, enzyme linked irnmunoabsorbent assay (ELISA), fluorescent immunosorbent assay (FIA), immunohistochemistry, chemical linked immunosorbent assay (CLIA), radioimmuno assay (RIA), flow cytometry (e.g., fluorescence activated ceil sorting or FACS), Western blot, and immunoblotting.
  • immunoassays for example, enzyme linked irnmunoabsorbent assay (ELISA), fluorescent immunosorbent assay (FIA), immunohistochemistry, chemical linked immunosorbent assay (CLIA), radioimmuno assay (RIA), flow cytometry (e.g., fluorescence activated ceil sorting or FACS), Western blot, and immunoblotting.
  • Additional applicable immunotechniques include competitive and noncompetitive assay systems, e.g., "sandwich” immunoassays, immunoprecipitation assays,
  • ELISAs include a number of variations.
  • the ELISA comprises preparing a target antigen, coating the wells of a muftiwelf microliter plate or other matrix material with the antigen, adding primary antibody, and incubating for a period of time, followed by addition of labeled secondary antibody.
  • the BioPlexTM 2200 system can be employed with a target antigen bound to a fluoromagnetic bead with a distinct fluorescent signature.
  • An aliquot of a patient sample e.g. serum, plasma
  • Patient antibodies binding specifically to the target antigen are detected by a fluorophore-labeied secondary antibody.
  • Multiple bead classes e.g. different fluorescent signatures, with different target antigens, can be used simultaneously or multiplexed.
  • bead-based assays e.g., where an antibody may be bound to the bead for detecting an antigen in the patient sample.
  • a fluorophore-labeied secondary antibody recognizing the antigen-antibody complex would act as a detector in this sandwich-assay format.
  • the chimeric anti-dsDNA'chromatm antibodies described herein can be conjugated or otherwise associated with a detectable label.
  • the chimeric anti- dsDNA/chromatin antibody (primary antibody) is detected using a secondary antibody that is conjugated or associated with a detectable label.
  • the association can be direct e.g., a covalent bond, or indirect, e.g., using a secondary binding agent, chelator, or linker.
  • the terms "detectable agent,” “detectable label,” “detectable moiety,” “label,” “imaging agent,” and like terms are used synonymously herein.
  • both the primary and secondary antibodies are labeled, e.g., with the same or with different labels.
  • the label can include an optical agent such as a fluorescent agent, phosphorescent agent, chemiluminescent agent, etc.
  • an optical agent such as a fluorescent agent, phosphorescent agent, chemiluminescent agent, etc.
  • agents e.g., dyes, probes, labels, or indicators
  • Fluorescent agents can include a variety of organic and/or inorganic small molecules or a variety of fluorescent proteins and derivatives thereof.
  • fluorescent agents can include but are not limited to cyanines, phthalocyanines, porphyrins, indocyanines, rhodamines, phenoxazines, phenylxanthenes, phenothiazines, phenoselenazines, fluoresceins, benzoporphyrins, squaraines, dipyrrolo pyiimidones, tetracenes, quinolines, pyrazines, corrins, croconiums, acridones, phenanthridines, rhodamines, acridines, anthraquinones, chalcogenopyrylium analogues, chlorins, naphthalocyanines, methine dyes, indolenium dyes,
  • the presently disclosed antibodies can be used for immunoassays, e.g., Western blots, ELISAs, FACS, immunoprecipitation, immunohistochemistry, immunofluorescence (e.g., using cells or tissue from a cell line or patient sample).
  • immunoassays e.g., Western blots, ELISAs, FACS, immunoprecipitation, immunohistochemistry, immunofluorescence (e.g., using cells or tissue from a cell line or patient sample).
  • cells or cellular material used in the immunoassay is fixed. In some embodiments, cells or cellular material is not fixed.
  • a radioisotope can be used as a label, and can include radionuclides that emit gamma rays, positrons, beta and alpha particles, and X-rays. Suitable radionuclides include but are not limited to 225 Ac, 72 As, 211 At, ! 1 B, 128 Ba, 212 Bi, 75 Br, 77 Br, 14 C, 109 Cd, 62 Cu, 64 Cu,
  • the antibody can be associated with a secondary binding figand or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate.
  • suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase (HRP) and glucose oxidase.
  • Secondary binding ligands include, e.g., biotin and avidin or streptavidin, as known in the art.
  • the label is a fluorescent protein sequence, and can be recombinantfy combined with the antibody polypeptide sequence.
  • Antibodies are generally labeled i an area that does not interfere with target binding, or in this case, with stability of the immune complex.
  • the detectable moiety is attached to the constant region, or outside the CDRs in the variable region.
  • the optimal position for attachment may be located elsewhere o the antibody, so the position of the detectable moiety can be adjusted accordingly.
  • the ability of the antibody to associate with the epitope is compared before and after attachment to the detectable moiety to ensure that the attachment does not unduly disrupt binding.
  • the presently described chimeric anti-dsDNA/chromatin antibodies typically bind to the target (dsDNA or chromatin) with a binding affinity of about 10 6 , 10 ', 10 8 , 10 9 , 10 i0 , 10 1 1 , or 10 lji M “1 (e.g., with a Kd in the micromolar (10 °), nanomolar (10*), picomolar (10 " :2 ), or lower range).
  • the affinity of the chimeric and- dsDNA/chromatin antibody for its target will be similar to native antibodies generated against the same target (e.g., autoantibodies generated against dsDNA or chromatin).
  • the affinities will be similar, e.g., within one order of magnitude.
  • the affinity is expressed in terms of Kd, wherein
  • Kd [antibody] x [target]/ [antibody-target complex].
  • the "antibody” in the above equation can refer to a chimeric antibody as described herein
  • the "target” can refer to dsDNA or chromatin
  • the antibody-target complex can refer to a complex comprising the chimeric antibody bound to dsDNA or chromatin.
  • Kd reduced dissociation
  • the specificity of antibody binding can be defined in terms of the comparative dissociation constants (Kd) of the antibody for the target as compared to the dissociation constant with respect to the antibody and other materials in the environment or unrelated molecules in general.
  • Kd comparative dissociation constants
  • the Kd for the antibody with respect to the unrelated material will be at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200- fold or higher than Kd with respect to the target.
  • A. targeting moiety will typically bind with a Kd of less than about 1000 nM, e.g., less than 250, 100, 50, 20 or lower nM, In some embodiments, the Kd of the affinity agent is less than 15, 10, 5, or I M. In some embodiments, the Kd is I - 100 nM, 0.1-50 nM, 0.1-10 nM, or 1-20 n M.
  • the value of the dissociation constant (Kd) can be determined by well- known methods, and can be computed even for complex mixtures by methods as disclosed, e.g., in Caceci el al. Byte (1984) 9:340-362.
  • Affinity of an antibody, or any targeting agent, for a target can be determined according to methods known in the art, e.g., as reviewed in Ernst et al. Determination of Equilibrium Dissociation Constants, Therapeutic Monoclonal Antibodies (Wiley & Sons ed, 2009).
  • ELISA Enzyme linked immunosorbent signaling assay
  • an antibody specific for target of interest is affixed to a substrate, and contacted with a sample suspected of containing the target. The surface is then washed to remove unbound substances.
  • Target binding can be detected in a variety of ways, e.g., using a second step with a labeled antibody, direct labeling of the target, or labeling of the primary antibody with a label that is detectable upon antigen binding.
  • the antigen is affixed to the substrate (e.g., using a substrate with high affinity for proteins, or a Strepavidin-biotin interaction) and detected using a labeled antibody (or other targeting moiety).
  • a labeled antibody or other targeting moiety.
  • the Kd, Kon, and Koff can also be determined using surface plasmon resonance (SP ).
  • SP surface plasmon resonance
  • Binding affinity can also be determined by anchoring a biotinylated interactant to a streptaviden (SA) sensor chip. The other interactant is then contacted with the chip and detected, e.g., as described in Abdessamad et al. (2002) Nuc. Acids Res. 30:e45.
  • SA streptaviden
  • Binding affinity can also be determined using comparative methods. For example, a set of components with known affinities can be compared to the test components (i.e. , antibody and target) under various conditions, e.g., wash conditions of various stringencies.
  • the genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody.
  • Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby, Immunology (3 rd ed. 1997)).
  • the expression system is a mammalian cell expression, such as a hybridoma, or a CHO cell expression system. Many such systems are widely available from commercial suppliers.
  • the heavy and light chains can be expressed using a single vector, e.g., in a di-cistronic expression unit, or under the control of different promoters. In other embodiments, the heavy and light chains can be expressed using separate vectors, or can be expressed in different cells and later combined.
  • the presence of such autoantibodies in a patient sample is indicative of certain autoimmune conditions including systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), Sjogren's syndrome (SS), scleroderma (systemic sclerosis), dermatomyositis (DM), polymyositis (PM), CREST syndrome.
  • SLE systemic lupus erythematosus
  • MCTD mixed connective tissue disease
  • SS Sjogren's syndrome
  • SS scleroderma
  • DM dermatomyositis
  • PM polymyositis
  • CREST syndrome CREST syndrome.
  • Autoantibodies specific for dsDNA and/or chromatin are also found in rheumatoid arthritis, Felty's syndrome, and juvenile arthritis.
  • a review of anti-dsDNA and anti-chromatin related disorders include Kavanaugh et ah (2002) Arthritis &. R
  • chimeric anti-dsDNA/chromatin antibodies can be included in a kit.
  • the chimeric anti-dsDNA/chromatin antibody is provided in a known amount and packaged, e.g., for shipping and storage (e.g., lyophilized, or in a buffer).
  • the kit can be designed for calibrating the binding or affinity of test antibodies specific for dsDNA and/or chromatin, e.g., native antibodies of known specificity or antibodies from a sample that may or may not include antibodies specific for dsDNA and/or chromatin.
  • the kit will include appropriate instructions to prepare a calibration curve using a chimeric anti- dsDNA/chromatin antibody as described herein, or include multiple containers of the chimeric anti-dsDNA'chromatin antibody at appropriate dilutions to prepare a calibration curve.
  • the antibody included in the kit is labeled (directly or indirectly).
  • the kit includes a secondary antibody (e.g., detectably labeled) specific for the chimeric anti-dsDNA/chromatin antibody.
  • the secondary antibody is specific for both the chimeric anti-dsDNA/chromatin antibody and the intended test antibody. In some embodiments, more than one secondary antibody is included with the kit.
  • the kit includes at least one tube or other container with a known amount of dsDNA. In some embodiments, the kit includes at least one tube or other container with a known amount of chromatin. In some embodiments, the dsDNA and/ or chromatin is detectably labeled or attached to a matrix.
  • the kit includes a chimeric anti-dsDNA/chromatin antibody as described herein and additional antibodies specific for different antigens.
  • the kit is designed for calibrating multiple antibodies with different targets.
  • the kit can include a chimeric anti-dsDNA/chromatin antibody as described herein, and at least one additional antibody specific for a different target, e.g., an autoimmune target such as other nuclear components.
  • the kit includes a known amount of the at least one additional antibody, packaged as described above for the anti- dsDNA/chromatin antibody.
  • the at least one additional antibody targets a nuclear or nucleolar antigen, e.g., an antigen selected from the group consisting of ribosomal protein, SS-A52, SS-A60, SS-B, 8m, Sm/RNP, RNP-A, RNP-68, Scl-70, Jo-1 and centromere B.
  • the kit can include any 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 1 additional antibodies in any combination. [01 ⁇ 7]
  • the kit includes at least one tube or other container with a known amount of the target of the at least one additional antibody.
  • the target of the at least one additional antibody is detectably labeled or attached to a matrix.
  • the kit includes a chimeric anti-dsDNA/chromatin antibody has a constant region from a human antibody and labeled secondary antibody specific for human antibodies (e.g., goat anti-human, rabbit anti-human, rat anti-human, etc.).
  • the kit includes at least one additional antibody with a different target specificity, wherein the at least one additional antibody is recognized by the same secondary antibody as the anti-dsDNA/chromatin antibody.
  • the at least one additional antibody is recognized by a different secondary antibody, e.g., labeled with a different label.
  • the at least one additional antibody has a constant region from a human antibody.
  • the at least one additional antibody can either be a native antibody (e.g., derived from a human sample), a recombinantly produced antibody, or a chimeric antibody.
  • Antibody candidates were selected after initial testing for accelerated stability studies. The antibodies were recombinantlv expressed and separated using Protein A column. Elution was carried out using Glycine-HCl Glycerol (highest yield and titer) or

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Abstract

La présente invention concerne des anticorps permettant de déterminer la concentration d'anticorps anti-ADNdb et anti-chromatine dans des échantillons biologiques.
EP13834463.5A 2012-09-05 2013-09-05 Anticorps chimère anti-adndb/anti-chromatine Withdrawn EP2893041A4 (fr)

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